Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility

Abstract

Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.

Fig. 1. The galactan and the mycobacterial cell envelope.

(A) Schematic depiction of the envelope of mycobacteria highlighting the mAGP (mycolyl-arabinogalactan-peptidoglycan). The glycans are depicted with the standard nomenclature. (B) Galactofuranosyl transferase 2 (GlfT2) extends the galactan to its final length. Polymerase activity can be probed in vitro using purified protein, synthetic acceptor, and sugar donor. (C) Overlayed matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) spectra produced by GlfT2 metal ion binding variants. Number of Galf residues added to acceptor noted above major product peaks.

Fig. 2. Impact of galactan truncation on fitness.

(A) Growth rate monitored in standard 7H9 medium. (B) RNA-seq data highlighting genes substantially up-regulated in D267E_comp cells during hyperosmotic challenge. (C) Changes in the minimum inhibitory concentrations (MICs) of hydrophobic drugs (green), like novobiocin, versus hydrophilic drugs (black), like streptomycin. Antibiotic targets are listed.

Fig. 3. Galactan chain length affects cell shape and periplasm size.

(A) Time-lapse microscopy images of M. smegmatis glfT2 mutants. Galactan truncation led to blebbing morphology (arrows) and, in multiple instances, cell rupture during division. (B) TEM of M. smegmatis glfT2 mutants highlighting the periplasm (arrows). (C) Galactan truncation decreases periplasm size in M. smegmatis (P < 0.0001). (D) Introduction of glfT2 orthologs affords access to strains with altered galactan chain length. (E) TEM images were used to quantify periplasm thickness across ortholog complemented strains. (F) Periplasm size and galactan chain length correlate linearly.

Fig. 4. Native-state imaging of glfT2 engineered M. smeg.

(A) Cryo-TEM micrographs of WT, WT_comp, and D267E_comp strains. Boundaries of periplasm are highlighted with red lines (left). (B) Quantification of periplasm size. (C) Cryo-TEM data analysis correlates with galactan chain length and periplasm size.